Phosphorylated matrix noncollagenous proteins (matrix phosphoproteins or MPP) have been proposed to play an important role in initiating the mineralization mechanism of bone and dentin. In particular, the phosphate groups of MPP have been demonstrated to be specifically involved in calcium ion binding. The addition of such phosphates to MPP during core-protein processing may be catalyzed by a novel membrane-bound enzyme, named matrix phosphoprotein kinase (MPP kinase). The goal of the present proposal is to elucidate the molecular mechanism of matrix protein phosphorylation through the following studies: (1) Purification of MPP kinase from a homogeneous population of rat osteosarcoma ROS 17/2.8 osteoblast-like cells. To solubilize MPP kinase from microsomal membranes, different detergents will be screened to assess the maximum kinase activation. The solubilized kinase will be purified by different chromatographic procedures. To characterize MPP kinase, optimum working conditions and enzyme kinetics will be examined. (2) Determination of the substrate-, phosphoamino acid residue-, and sequence-specificities for MPP kinase. To distinguish MPP kinase from other kinases, the kinetics differences (i.e., different kinases on same substrate) and co-substrate specificity (i.e., same kinase on different substrates) will be investigated. The preferential phosphoamino acid residue (phosphoserine or phosphothreonine) and the sequence specificity among the sites of the phosphorylated substrates will also be determined. (3) Regulation of MPP kinase by vitamin D3 and transforming growth factor-beta through quantitation of MPP kinase and changes in kinetics at different time course of enzyme activation. Vitamin D and transforming growth factor-beta as extracellular signals will first be added to cell culture medium to regulate intracellular activity and cell extract will then be subjected to further investigation. A polyclonal antibody against MPP kinase will be raised and Western blot and immunoprecipitation will be used to measure the amount of MPP kinase present after stimulation. These data will provide vital information regarding the significance of matrix protein phosphorylation in matrix-mediated mineralization of bone and dentin.